Proteases of the subtilisin type (subtilases, subtilopeptidases, EC 3.4.21.62), in particular subtilisins, are classed as belonging to the serine proteases, owing to the catalytically active amino acids. They are naturally produced and secreted by microorganisms, in particular by Bacillus species. They act as unspecific endopeptidases, i.e. they hydrolyze any acid amide bonds located inside peptides or proteins. Their pH optimum is usually within the distinctly alkaline range. A review of this family is provided, for example, by the paper “Subtilases: Subtilisin-like Proteases” by R. Siezen, pages 75 95 in “Subtilisin enzymes”, edited by R. Bott and C. Betzel, New York, 1996. Subtilisins are suitable for a multiplicity of possible technical uses, as components of cosmetics and, in particular, as active ingredients of detergents or cleaning agents.
Enzymes are established active ingredients of washing and cleaning products. In this connection, proteases bring about the breakdown of proteinaceous soilings on the material to be cleaned such as, for example, textiles or hard surfaces. In favorable cases there are synergistic effects between the enzymes and the other constituents of the relevant products. This is described for example in U.S. Pat. No. 6,008,178. Owing to their favorable enzymic properties such as stability or pH optimum, subtilisins stand out among the washing and cleaning product proteases. The most important ones and the most important strategies for their technical development are stated below.
The development of washing product proteases is based on natural enzymes which are preferably produced by microbes. They are optimized by mutagenesis methods known per se, for example point mutagenesis, deletion, insertion or fusion with other proteins or protein portions or via other modifications for use in washing and cleaning products.
Thus, for example, according to application WO 93/07276, the protease 164-A1 which is obtainable from Bacillus spec. 164 A1 and is supplied by Chemgen Corp., Gaithersburg, Md., USA, and Vista Chemical Company, Austin, Tex., USA, is suitable for use in washing and cleaning products. Other examples are the alkaline protease from Bacillus sp. PD138, NCIMB 40338 of Novozymes (WO 93/18140), the proteinase K 16 derived from Bacillus sp. ferm. BP 3376 of Kao Corp., Tokyo, Japan, (U.S. Pat. No. 5,344,770) and, according to WO 96/25489 (Procter & Gamble, Cincinnati, Ohio, USA), the protease from the psychrophilic organism Flavobacterium balustinum. Further proteases of microbial origin which are suitable for use in washing and cleaning products are also known from the patent literature: for example from Pseudomonas (WO 00/05352), from Metarrhizium (EP 601005), from Bacillus alkalophilus DMS 6845 or DSM 5466 (DE 4411223) and various other microorganisms (WO 95/07350, EP 1029920, EP 578712, WO 01/00764, U.S. Pat. No. 6,197,740, WO 01/16285).
Subtilisin BPN′ which is derived from Bacillus amyloliquefaciens, and B. subtilis, respectively, has been disclosed in the studies by Vasantha et al. (1984) in J. Bacteriol., Volume 159, pp. 811 819 and by J. A. Wells et al. (1983) in Nucleic Acids Research, Volume 11, pp. 7911 7925. Subtilisin BPN′ serves as reference enzyme of the subtilisins, in particular with respect to numbering of positions. The application CA 2049097 discloses multiple mutants of this molecule, especially in relation to their stability in washing and cleaning products. Variants which are obtained by point mutations in the loop regions of this enzyme and which have reduced binding to the substrate with, at the same time, an increased rate of hydrolysis are presented for example in the patent applications WO 95/07991 and WO 95/30010. Washing products with such BPN′ variants are disclosed for example in the patent application WO 95/29979.
The publications by E. L. Smith et al. (1968) in J. Biol. Chem., Volume 243, pp. 2184 2191, and by Jacobs et al. (1985) in Nucl. Acids Res., Volume 13, pp. 8913-8926 introduce the protease subtilisin Carlsberg. It is naturally produced by Bacillus licheniformis and was and, respectively, is obtainable under the trade name Maxatase® from Genencor International Inc., Rochester, N.Y., USA, and under the trade name Alcalase® from Novozymes A/S, Bagsvaerd, Denmark. Variants thereof which are obtainable by point mutations and have reduced binding to the substrate with a simultaneously increased rate of hydrolysis are disclosed, for example, by the application WO 96/28566 A2. These are variants in which single or multiple substitutions in the loop regions of the molecule have been carried out.
The protease PB92 is produced naturally by the alkaliphilic bacterium Bacillus nov. spec. 92 and was obtainable under the trade name Maxacal® from Gist-Brocades, Delft, The Netherlands. Its original sequence is described in patent application EP 283075 A2. Variants of said enzyme which have been obtained by point mutation and which are suitable for use in detergents and cleaning agents are disclosed in the applications WO 94/02618 and EP 328229, for example.
The subtilisins 147 and 309 are sold by Novozymes under the trade names Esperase® and Savinase®, respectively. They are originally derived from Bacillus strains disclosed by the application GB 1243784. Variants of said enzymes, which have been developed by means of point mutagenesis with respect to usage in washing and cleaning products, are disclosed, for example, in the applications WO 94/02618 (see above), WO 89/06279, WO 95/30011 and WO 99/27082. The application WO 89/06279 aimed at achieving higher oxidation stability, an increased rate of proteolysis and enhanced washing performance. It reveals that substitutions at particular positions alter the physical or chemical properties of subtilisin 147 or 309 molecules. The application WO 95/30011 introduces variants of subtilisin 309 which have point mutations in the loop regions of the molecule and thus exhibit reduced adsorption to the substrate with a simultaneously increased rate of hydrolysis. The application WO 99/27082 develops variants of, by way of example, subtilisin 309, whose washing performance is enhanced by enlarging the active loops by inserting at least one amino acid.
The B. lentus alkaline proteases are highly alkaline proteases from Bacillus species. The wild-type enzyme is derived from an alkaliphilic bacillus strain and itself shows comparatively high stability towards oxidation and the action of detergents. This strain was, according to the application WO 91/02792 (EP 493398 and U.S. Pat. No. 5,352,604), deposited under the number DSM 5483. According to the same application, the enzyme can be expressed heterologously in the host Bacillus licheniformis. Its three-dimensional structure is described in the publication of Goddette et al. (1992), J. Mol. Biol. Volume 228, pages 580-595: “The crystal structure of the Bacillus lentus alkaline protease, Subtilisin BL, at 1.4 Å resolution”. Variants of this enzyme which can be obtained by point mutation and are suitable for use in washing and cleaning products are disclosed in WO 92/21760 (U.S. Pat. No. 5,340,735, U.S. Pat. No. 5,500,364 and U.S. Pat. No. 5,985,639) and WO 95/23221 (U.S. Pat. No. 5,691,295, U.S. Pat. No. 5,801,039 and 5,855,625). The strategy underlying WO 95/23221, namely deliberate alteration of the charge conditions near the substrate binding pocket is explained in U.S. Pat. No. 6,197,589. Further variants of this protease are described in the as yet unpublished applications DE 10121463 and DE 10153792.
Subtilisin DY has originally been described by Nedkov et al. 1985 in Biol. Chem Hoppe-Seyler, Volume 366, pp. 421-430. According to the application WO 96/28557, for example, it may be optimized via specific point mutations in the active loops for usage in detergents and cleaning agents, producing variants having reduced adsorption and an increased rate of hydrolysis.
The enzyme thermitase which is to be assigned to the subtilases, but no longer to the subtilisins, (cf. R. Siezen, pages 75-95 in “Subtilisin enzymes”, published by R. Bott and C. Betzel, New York, 1996) and is produced naturally by Thermoactinomyces vulgaris was originally described by Meloun et al. (FEBS Lett. 1983, pp. 195-200). The application WO 96/28558, for example, discloses variants having reduced absorption and an increased rate of hydrolysis, owing to substitutions in the loop regions. However, thermitase is a molecule whose sequence overall deviates considerably from those of the other subtilisins.
Proteinase K is also a subtilase which has comparatively low homology, for example, to B. lentus alkaline protease. Proteinase K is originally from the microorganism Tritirachium album Limber and has been described by K. D. Jany and B. Mayer 1985 in Biol. Chem. Hoppe-Seyler, Vol. 366, pp. 485-492. The application WO 96/28556 discloses numerous variants of proteinase K which are obtainable by point mutagenesis and have reduced adsorption to the substrate and an increased rate of hydrolysis.
WO 88/07581, finally, discloses the very similar proteases TW3 and TW7, inter alia for usage in washing and cleaning products.
The applications EP 199404, EP 251446, WO 91/06637 and WO 95/10591, for example, describe further proteases which are suitable for technical use, in particular in detergents and cleaning agents. The proteases of the application EP 199404 are various BPN′ variants which are based on the patent EP 130756. EP 251446 discloses numerous BPN′ variants, obtainable by exchanging individual amino acids. The proteases of the application WO 91/06637 are distinguished by point mutations of BPN′ in positions 123 and/or 274. WO 95/10591 reveals variants, mainly of the Bacillus lentus protease, which have mutations in position 76 and also other positions.
Other known proteases are, for example, the enzymes obtainable under the trade names Durazym®, Relase®, Everlase®, Nafizym, Natalase®, Kannase® and Ovozymes® from Novozymes, under the trade names Maxapem®, Purafect®, Purafect OxP® and Properase® from Genencor, under the trade name Protosol® from Advanced Biochemicals Ltd., Thane, India and under the trade name Wuxi® from Wuxi Snyder Bioproducts Ltd., China.
One strategy for enhancing the washing performance of subtilisins is to replace randomly or specifically individual amino acids by others in the known molecules, and to test the variants obtained for their washing performance contributions. This strategy is pursued by some of the further developments indicated in each case above, for example EP 130756. The allergenicity of the enzymes can also be improved for example according to WO 99/49056, WO 99/49057 and WO 01/07575 with certain amino acid exchanges or deletions.
In order to enhance the washing performance of subtilisins, numerous applications pursued the strategy of inserting additional amino acids into the active loops, thus, for example, apart from the already mentioned WO 99/27082, also the applications published with the numbers WO 00/37599, WO 00/37621 to WO 00/37627 and WO 00/71683 to WO 00/71691. Said strategy should accordingly be applicable in principle to all subtilisins belonging to either of the subgroups I S1 (true subtilisins) or I S2 (highly alkaline subtilisins).
Another strategy of enhancing the performance is to modify the surface charges and/or the isoelectric point of the molecules, thereby altering their interaction with the substrate. Variations of this kind are disclosed, for example, by U.S. Pat. No. 5,665,587 and the applications EP 405901, EP 945502 A1, WO 91/00334 and WO 91/00345. WO 92/11348 discloses point mutations for reducing the pH-dependent variation in the molecular charge. The application WO 00/24924 derives from this principle a method for identifying variants which are supposedly suitable for usage in washing and cleaning products; all variants disclosed here have at least one substitution at position 103, preference being given to multiple variants containing no substitution relevant to the present application. According to WO 96/34935, it is also possible to increase the hydrophobicity of the molecules for the purpose of enhancing the performance in washing and cleaning products, and this may influence the stability of the enzyme.
The application WO 99/20727 discloses subtilisin variants as are obtained by a method of the application WO 00/24924: they all comprise at least one substitution at position 103, combined with a multiplicity of other possible substitutions. The applications WO 99/20723 and WO 99/20726 disclose the same mutants for washing and cleaning products which additionally contain an amylase, or bleach.
Another method for modulating the efficiency of proteases is to form fusion proteins. Thus, for example, the applications WO 98/13483 and WO 00/01831 disclose fusion proteins composed of proteases and an inhibitor such as the Streptomyces subtilisin inhibitor. Another possibility is, for example according to WO 97/28243 or WO 99/57250, to couple to the cellulose binding domain (CBD), which is derived from cellulases, to increase the concentration of active enzyme in the direct vicinity of the substrate. According to WO 99/48918 the allergenicity or immunogenicity is reduced by coupling a peptide linker, and polymers thereon.
Variants with improved performance due to randomly generated amino acid exchanges and subsequent selection are revealed for example in WO 99/20769. A random method, based on the phage display system, for evolving proteases for use in washing and cleaning products is revealed for example in the application WO 97/09446.
A modern direction in enzyme development is to combine, via statistical methods, elements from known proteins related to one another to give novel enzymes having properties which have not been achieved previously. Methods of this kind are also listed under the generic term directed evolution and include, for example, the following methods: the STEP method (Zhao et al. (1998), Nat. Biotechnol., Volume 16, pp. 258-261), random priming recombination (Shao et al., (1998), Nucleic Acids Res., Volume 26, pp. 681-683), DNA shuffling (Semmer, W. P. C. (1994), Nature, Volume 370, pp. 389-391) or recursive sequence recombination (RSR; WO 98/27230, WO 97/20078, WO 95/22625) or the RACHITT (Coco, W. M. et al. (2001), Nat. Biotechnol., Volume 19, pp. 354-359). A survey of such methods is also provided by the prior article “Gerichtete Evolution und Biokatalyse” by Powell et al. (2001), Angew. Chem., Vol. 113, pages 4068-4080.
Another, in particular complementary, strategy is to increase the stability of the proteases concerned and thus to increase their efficacy. For example, U.S. Pat. No. 5,230,891 has described stabilization via coupling to a polymer for proteases used in cosmetics; said stabilization is accompanied by enhanced skin compatibility. Especially for detergents and cleaning agents, on the other hand, stabilizations by point mutations are more familiar. Thus, according to U.S. Pat. No. 6,087,315 and U.S. Pat. No. 6,110,884, it is possible to stabilize proteases by replacing particular tyrosine residues with other residues. WO 89/09819 and WO 89/09830 describe relatively thermostable BPN′ variants obtained by amino-acid substitution. Other possible examples of stabilization via point mutagenesis, which have been described, are:    replacing particular amino acid residues with proline according to WO 92/19729, and, respectively, EP 583339 and U.S. Pat. No. 5,858,757 and according to EP 516200;    introducing more polar or more highly charged groups on the molecule surface, according to EP 525610, EP 995801 and U.S. Pat. No. 5,453,372;    enhancing the binding of metal ions, in particular via mutagenesis of calcium binding sites, for example according to the teaching of the applications WO 88/08028 and WO 88/08033;    blocking autolysis by modification or mutagenesis, for example according to WO 98/20116 or U.S. Pat. No. 5,543,302.    Combination of a plurality of stabilization strategies as disclosed in the application EP 398539 A1.    According to U.S. Pat. No. 5,340,735, U.S. Pat. No. 5,500,364, U.S. Pat. No. 5,985,639 and U.S. Pat. No. 6,136,553, the positions relevant to stabilization can be found by analysis of the three-dimensional structure.
The documents EP 755999 and WO 98/30669, for example, disclose that proteases may be used together with α-amylases and other washing product enzymes in order to enhance the washing or cleaning performance. For example, EP 791046 discloses the possibility of combination with lipases. The application WO 95/10592 for example reveals that the variants previously described in WO 95/10591 for use in washing products are also suitable for use in bleaches. U.S. Pat. No. 6,121,226 for example discloses the simultaneous use of protease and soil release agents in washing products.
The application WO 97/07770 for example discloses that some proteases established for use in washing products are also suitable for cosmetic purposes. A further possible technical use of proteases is presented for example in the application EP 380362 A1. This relates to organic chemical syntheses, and the subtilisins said according to this application to be suitable for this are those stabilized by point mutagenesis.
The diverse technical areas of use presented here by way of example require proteases with different properties relating for example to the reaction conditions, the stability or the substrate specificity. Conversely, the possibilities of technical use of proteases, for example in the context of a washing or cleaning product formula, depend on further factors such as stability of the enzyme towards high temperatures, towards oxidizing agents, its denaturation by surfactants, on folding effects or on desired synergies with other ingredients.
Thus, there continues to be a great need for proteases which can be employed technically and which, owing to the large number of their areas of application, in their totality cover a wide range of properties, including very subtle differences in performance.
The basis for this is expanded by novel proteases which in turn are capable of further development targeted at specific areas of application.